Polycistronic expression systems in insects can be used for applications such as recombinant protein production in cells, enhanced transgenesis methods, and the development of novel pest-control strategies based on the sterile insect technique (SIT). Here we tested the performance of four picornaviral 2A self-cleaving peptides (TaV-2A, DrosCV-2A, FMDV 2A1/31 and FMDV 2A1/32) for the co-expression and differential subcellular targeting of two fluorescent marker proteins in cell lines (Anastrepha suspensa AsE01 and Drosophila melanogaster S2 cells). We found that all four 2A peptides showed comparable activity in cell lines, leading to the production of independent upstream and downstream proteins that were directed to the nucleus or membrane by a C-terminal nuclear localization signal (NLS) on the upstream protein and a poly-lysine/CAAX membrane anchor on the downstream protein. TaV-2A and DrosCV-2A were inserted into piggyBac constructs to create transgenic D. suzukii strains, confirming efficient ribosomal skipping in vivo. Interestingly, we found that the EGFP-CAAX protein was distributed homogeneously in the membrane whereas the DsRed-CAAX protein formed clumps and aggregates that induced extensive membrane blebbing. Accordingly, only flies expressing the DsRed-NLS and EGFP-CAAX proteins could be bred to homozygosity whereas expression of EGFP-NLS and DsRed-CAAX was lethal in the homozygous state. Our results therefore demonstrate that the 2A constructs and two novel targeting motifs are functional in D. suzukii, and that the combination of EGFP-NLS and DsRed-CAAX shows dosage-dependent lethality. These molecular elements could be further used to improve expression systems in insects and generate novel pest control strains.

中文翻译：

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使用picornaviral 2A肽在昆虫细胞和铃木果蝇中双顺反子表达和蛋白质的差异定位。

昆虫中的多顺反子表达系统可用于多种应用，例如细胞中重组蛋白的生产，增强的转基因方法以及基于无菌昆虫技术（SIT）的新型害虫控制策略的开发。在这里，我们测试了四种皮甲病毒2A自裂解肽（TaV-2A，DrosCV-2A，FMDV 2A1 / 31和FMDV 2A1 / 32）在细胞系中两种荧光标记蛋白的共表达和差异亚细胞靶向的性能（ Anastrepha suspensa AsE01和Drosophila melanogaster S2细胞。我们发现所有4种2A肽在细胞系中均显示出可比的活性，导致产生独立的上游和下游蛋白质，这些蛋白质通过上游蛋白质上的C端核定位信号（NLS）和下游蛋白质上的聚赖氨酸/ CAAX膜锚定物定向到细胞核或膜。将TaV-2A和DrosCV-2A插入piggyBac构建体中以创建转基因铃木D.株，从而证实了体内有效的核糖体跳跃。有趣的是，我们发现EGFP-CAAX蛋白均匀地分布在膜中，而DsRed-CAAX蛋白形成团块和聚集体，引起大量膜起泡。因此，只有表达DsRed-NLS和EGFP-CAAX蛋白的果蝇才能被纯合，而EGFP-NLS和DsRed-CAAX的表达在纯合状态下是致​​命的。因此，我们的结果证明了2A构建体和两个新颖的靶向基序在铃木D.中具有功能，并且EGFP-NLS和DsRed-CAAX的组合显示出剂量依赖性致死性。这些分子元素可进一步用于改善昆虫的表达系统并产生新的害虫控制菌株。